Anti-Oxidant and Anti-Inflammatory Activities of Leaves of Barringtonia Racemosa
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Journal of Medicinal Plants Research pp. 095-102, December 2007 Available online http://www.academicjournals.org/JMPR ISSN 1996-0875©2007 Academic Journals Full Length Research Paper Anti-oxidant and anti-inflammatory activities of leaves of Barringtonia racemosa Mandana Behbahani, Abdul Manaf Ali, Radzali Muse and Noorjahan Banu Mohd Department of Cell and Molecular Biology, UPM, Malaysia. Accepted 17, July 2007 The medicinal plant of Barringtonia racemosa (lecythidaceae family) has been used widely in traditional medicine for anti-inflammation and anticancer in Malaysia. The present investigation was carried out to study of the anti-oxidant and anti-inflammatory effects of fully expanded leaf extracts of B. racemosa. Antioxidant activity was measured by using FTC, TBA and DPPH free radical scavenging methods and Griess assay was also used for the measurement of nitric oxide inhibition in lipopolysaccharide (LPS) and interferon- (IFN- )-treated RAW 264.7 cells. Different crude extracts of fully expanded leaf extracts of B. racemosa have exhibited a very good level of nitric oxide (NO) inhibitory and antioxidant activities. In the Griess assay, non polar extracts such as chloroform and hexane extracts were found to be strong inhibitors of NO at different concentrations (25, 50, 100 and 200 µg/ml) in comparison with polar extract (ethanol extract). Chloroform extract didn’t show cytotoxicity against RAW 264.7 cells at different concentrations contrary to hexane and ethanol extracts. The chloroform and hexane extracts exhibited very strong antioxidant properties when compared to Vitamin E (a-tocopherol) in the FTC and TBA methods, the chloroform and hexane extracts exhibited the radical scavenging activity with an IC50 value of 54.29 and 63 µg/ml respectively. Results demonstrated that chloroform extract of B. racemosa leaf may have the potential to be used as anti-inflammation and anti-oxidant agents and it was revealed that the active compound in B. racemosa is lycopene. Key words: Barringtonia racemosa, anti-oxidant, anti-inflammatory. INTRODUCTION Barringtonia racemosa is a tropical higher plant and is a has anti-tumor property and toxicity in mice (Khan et al., member of the Lecythidaceae family. B. racemosa is a 2001). whilst the ethanol extract of the plant leaves dis- moderate sized evergreen tree found in the West Coast played cytotoxicity against the HelA (human cervila of India, Sundarbans, Assam and Andaman Islands and carcinoma) cell line with a IC50 value of 10 g/ml Malaysia. It is used as a traditional medicine in Malaysia (Mackeen et al., 1997).The aqueous extract isolated from and locally known as putat. Its fruits are used to treat the stem bark of B. racemosa has been displayed to have cough, asthma and diarrhea; the seeds are aromatic and antinociceptive and toxicological effect on rats (Thomas useful in treating colics and ophthalmic problems. Pre- et al., 2002) while an ethanol extract of the roots of B. vious studies on some Barringtonia species including racemosa provided two novel clerodane diterpenoid Barringtonia asiatica, Barringtonia acutangul and Barring- nasimalun A and B by NMR and MS analysis( Khan et tonia lanceolata have showed that most of the species al., 2000). possess medicinal properties (Grosvenor et al., 1995; B. racemosa well known in Malaysia as a traditional Khan and Omoloso, 2002). Other related genus of Bar- medicine has been shown to exhibit anti-oxidant and anti- ringtonia edulis also was reported to possess definitive inflammatory effects. In the recent past, there has been sterility and used to induce abortion (Bourdy and Waller, an increase in the use of plants as sources of natural 1992). anti-oxidants for the scavenging of free radicals. The Previous investigation on the bioactivity of B. racemosa latter are known to initiate a series of (oxygen robbing) has shown that the ethanol extract of B. racemosa barks chain reactions resulting in oxidative tissue damage and a wide range of degenerative diseases, such as cancer and a host of cardiovascular diseases (Galati and O'Brien, 2004). *Corresponding Author email: [email protected] Deraniyagala et al. (2003) reported that the aqueous 096 J. Med. Plant. Res. Table 1. Percentage of antioxidant activity of leaves of Barringtonia racemosa extracted with three different solvents (chloroform, ethanol and hexane). Antioxidant activity of Barringtonia racemosa (%) Tocopherolic extract Chloroform extract Hexane extract Ethanol extract FTC TBA FTC TBA FTC TBA FTC TBA 67.08±1.32 46.4±1.54 79.13±1.75 58±1.42 76.52±1.64 54.4±1.14 51,39±0.30 33±0.54 Derived leaves were separately extracted using chloroform, hexane and ethanol at room temperature. Each value represents the mean ± SD (n = 3). bark extract of B. racemosa have several bioactivities interferon – ( Heras et al., 2001). such as antinociceptive effect and this effect was media- ted through opioid mechanisms. Culture of RAW 264.7 Cell The literature survey revealed that there are no scientific studies carried out regarding antioxidant and The murine monocytic macrophage cell line RAW 264.7 was anti-inflammatory activity of the leaves of B. racemosa. purchased from American Type Culture Collection (ATCC, USA). Hence, the present study is focused to evaluate the This macrophase cell line was initially established from a tumor induced by Abelson murine leukemia virus in Balb/c mice (Raschke antioxidant and anti-inflammatory potentials of different et al., 1978). The RAW 264.7 cells were cultured in Dulbecco’s extracts of leaves of B. racemosa. Modified Eagle Media (DMEM) (containing 4 mM L-glutamine, 4.5 g/L glucose, 1.5 g/L (w/v) sodium bicarbonate, 50 U/ml (w/v) penicillin, 50 Ag/ml (w/v) streptomycin) with 10% foetal calf serum MATERIAL AND METHOD (FCS). The cells were cultured at 37°C with 5% CO2 and were split twice a week. The RAW 264.7 cells were semi-adherent cells, Plant material where some cells grew in suspension, some were loosely attached to the surface and others flatted out and attached to the flask. The Ten kg of the fresh fully expended leaves of B. racemosa were subcultures were prepared by scraping and the sub cultivation ratio collected from Herbal unit, at University Putra Malaysia, in was between 1:3 to 1:6 (culture: media). December 2005. Young leaves (10 – 15 cm long) as well as older leaves (15 – 25 cm long) were chosen. The petioles were removed. The leaves without petioles were washed under running water, air Griess assay for nitric oxide inhibitory activity dried and ground. Out of 10 kg of the fresh leaves of B. racemosa, 1.8 kg of the dried powder was obtained. The dried plants were Briefly, cells were seeded in 96-well tissue culture plates (1 × 106 stored in plastic bags and kept in a dark at room temperature (28 – cells/100 µl) and incubated for 24 h at 37ºC with 5% CO2. One 30°C). hundred µl of chloroform, hexane and ethanol extracts in 0.1% DMSO was then added separately and serially diluted to give final concentrations of 25, 50, 100 and 200 µg/ml (Table 1). Cells were Extraction of compound using different organic solvents then stimulated with 200 U/ml (w/v) of IFN-g and 10 µg/ml (w/v) LPS for another 17 h. The presence of nitrite, a stable oxidized The dried plant sample (60 g) was placed in a stopped conical flask product of NO, was determined in cell culture media by Griess and macerated with 500 ml of 98% chloroform (Merck, Germany) at reagent (1%, w/v) of sulfanamide and 0.1% (w/v) N-(1-naphtyl) room temperature (28 – 30°C) for 3 days with occasional stirring. ethylenediamine dihydrochloride in 2.5% (v/v, H PO ). One hundred This experiment has done three times. The solvent was then filtered 3 4 µl of cell culture supernatant was removed and combined with 100 and evaporated in a rotary evaporator (Somatco, Germany) under µl of Griess reagent in a 96-well plate followed by spectro- vacuum at 45°C. The residue was placed in oven (Napco, USA) at photometric measurement at 550 nm using a microplate reader. 40ºC until dry. The crude extract was stored in a well-closed Nitrite concentration in the supernatants was determined by container and protected from light and kept in a refrigerator at −4°C. comparison with a sodium nitrite standard curve. The amount of live The extraction was carried out on three separate occasions (n = 3). cells was detected by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl- The above procedure was repeated using 95% ethanol and 98% tetrazolium bromide (MTT) cytotoxicity assay and values given in hexane(Merck, Germany) as solvent instead of chloroform, again percent cell cytotoxicity. replicated on three occasions (n = 3). Out of 60 g of the dried plant sample of B. racemosa could be able to produce 15 g ethanol, 14 g hexane, 18 g chloroform extracts. Measurement of nitrite For determination of nitric oxide concentration, the stable conver- Anti-inflammatory activity sion product of nitric oxide, nitrite (NO2−) was measured using the Griess reagent (Chi et al., 2001). After 24 h incubation 50µl of Inflammation is characterized by the over production of nitric supernatant from each well of cell culture plates was transferred oxidide (NO). The inducible isoform of NO synthase (iNOS) is into 96-well microplates and equal volume of Griess reagent (1% directly responsible for the generation of NO. Selective inhibition of sulfanilamide, 0.1% (w/v) of N-(1-naphthyl)-ethyline diamine iNOS and its biosynthetic product NO, has been shown to suppress hydrochloride, 2.5% (v/v, H3PO4) was then added to the super- inflammation in a variety of inflammation states.